High-throughput gene silencing and mRNA expression analysis in hepatocyte sandwich cultures.

High-throughput gene silencing and mRNA expression analysis in hepatocyte sandwich cultures.
Curr Protoc Toxicol. 2013 Feb;Chapter 14:Unit 14.11
Authors: Hollingshead BD, Gauthier LM, Burdick AD
Abstract
Primary hepatocyte sandwich cultures are useful for a variety of research applications where maintenance of metabolic competency is essential. To ensure an optimal hepatocellular phenotype, cells are seeded on collagen-coated dishes and embedded with an overlay of Matrigel. This culturing condition makes gene silencing by traditional reagent-mediated transfection methods challenging. Here, an siRNA delivery method in primary mouse hepatocytes that allows cells to be cultured with Matrigel overlay is described. This method delivers >80% mRNA silencing with minimal alterations in cell viability. A 96-well format allows for high-throughput RNA processing and downstream quantitative PCR applications and reduces time and resources. This format is particularly useful when experiments requiring many different sampling conditions (such as pharmacologic dose-response curves) are required.
PMID: 23408193 [PubMed - indexed for MEDLINE]

Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research